Functional Analysis of Injectable Substance Treatment on Surgically Injured Rabbit Vocal Folds.

OBJECTIVES: The objective of this study was to evaluate the efficacy of immediate injection treatments of dexamethasone, hyaluronic acid (HA)/gelatin (Ge) hydrogel and glycol-chitosan solution on the phonatory function of rabbit larynges at 42 days after surgical injury of the vocal folds, piloting a novel ex vivo phonatory functional analysis protocol. METHODS: A modified microflap procedure was performed on the left vocal fold of 12 rabbits to induce an acute injury. Animals were randomized into one of four treatment groups with 0.1 mL injections of dexamethasone, HA/Ge hydrogel, glycol-chitosan or saline as control. The left mid vocal fold lamina propria was injected immediately following injury. The right vocal fold served as an uninjured control. Larynges were harvested at Day 42 after injection, then were subjected to airflow-bench evaluation. Acoustic, aerodynamic and laryngeal high-speed videoendoscopy (HSV) analyses were performed. HSV segments of the vibrating vocal folds were rated by three expert laryngologists. Six parameters related to vocal fold vibratory characteristics were evaluated on a Likert scale. RESULTS: The fundamental frequency, one possible surrogate of vocal fold stiffness and scarring, was lower in the dexamethasone and HA/Ge hydrogel treatment groups compared to that of the saline control (411.52±11.63 Hz). The lowest fundamental frequency value was observed in the dexamethasone group (348.79±14.99 Hz). Expert visual ratings of the HSV segments indicated an overall positive outcome in the dexamethasone treatment group, though the impacts were below statistical significance. CONCLUSION: Dexamethasone injections might be used as an adjunctive option for iatrogenic vocal fold scarring. An increased sample size, histological correlate, and experimental method improvements will be needed to confirm this finding. Results suggested a promising use of HSV and acoustic analysis techniques to identify and monitor post-surgical vocal fold repair and scarring, providing a useful tool for future studies of vocal fold scar treatments.

Characterizing Vocal Fold Injury Recovery in a Rabbit Model With Three-Dimensional Virtual Histology.

OBJECTIVES/HYPOTHESIS: In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. METHODS: A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. RESULTS: The MRI modality allowed visualization of injury location and morphological internal features with 100-µm spatial resolution. The CT modality provided a view of the injury defect surface with 12-µm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. CONCLUSIONS: Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1578-1587, 2021.

Multimodal virtual histology of rabbit vocal folds by nonlinear microscopy and nano computed tomography.

Human vocal folds (VFs) possess a unique anatomical structure and mechanical properties for human communication. However, VFs are prone to scarring as a consequence of overuse, injury, disease or surgery. Accumulation of scar tissue on VFs inhibits proper phonation and leads to partial or complete loss of voice, with significant consequences for the patient's quality of life. VF regeneration after scarring provides a significant challenge for tissue engineering therapies given the complexity of tissue microarchitecture. To establish an effective animal model for VF injury and scarring, new histological methods are required to visualize the wound repair process of the tissue in its three-dimensional native environment. In this work, we propose the use of a combination of nonlinear microscopy and nanotomography as contrast methods for virtual histology of rabbit VFs. We apply these methods to rabbit VF tissue to demonstrate their use as alternatives to conventional VF histology that may enable future clinical studies of this injury model.